Sodium-independent transporter carrying acidic amino acid and its gene

ABSTRACT

It is intended to provide a sodium-independent transporter carrying an acidic amino acid and its gene. A protein having the amino acid sequence represented by SEQ ID NO: 1 and being capable of sodium-independently transporting an acidic amino acid and its analogs; a gene encoding this protein; a fused protein of the above protein with an auxiliary factor enabling the expression of its function; a gene encoding the same; a method of analyzing the function of a transporter using the same: and utilization thereof.

TECHNICAL FIELD

The present invention relates to a protein associated with thesodium-independent transport of acidic amino acids and its analogue,fusion protein thereof, as well as a gene encoding said protein. Thepresent invention also relates to a method for controlling the cellproliferation or for altering the in vivo pharmacokinetics of apharmaceutical, toxic substance or xenobiotics by modulating an abilityto transport acidic amino acids and its analogue possessed by a proteinassociated with the sodium-independent transport of acidic amino acidsand its analogue, by means of employing said protein, its fusionprotein, its specific antibody, or its function-promoting substance orfunction-suppressing substance, as well as an agent for controlling anability to transport acidic amino acids and its analogue comprising saidsubstances.

BACKGROUND ART

A cell always requires the uptake of an amino acid as a nutrition, andsuch a function is exerted by an amino acid transporter which is amembrane protein existing in a cell membrane. The amino acid transporteris distributed in a specific site in each tissue in a multicellularorganism and plays an important role in expressing the specific functionof each tissue. For example, in kidney cells and small intestine, itplays a role for epithelial absorption of amino acid in lumen and, innerve tissues, it is in charge of recovery of amino acid as aneurotransmitter released as a result of neurotransmission and also ofsupply of amino acid as a precursor for neurotransmitter to nerve cells.Further, it exists in blood-brain barrier and placental barrier andmakes permeation of the amino acid possible.

With regard to an amino acid transport mechanism, its identification andclassification have been conducted using cultured cells and membranespecimens since 1960's and, reflecting the multiplicity of amino acidmolecules, many transport systems have been described. However, therehas been no independent transport system for each amino acid but most ofthe amino acid transports have been conducted by a few kinds oftransport systems which transport several amino acids having similarside chains (Christensen, Physiol. Rev., volume 70, page 43, 1990).

Transport of acidic amino acids such as glutamic acid and aspartic acidhaving carboxyl group on a side chain has been believed to be carriedout by both of a sodium-dependent transporter which requires sodium ionfor its function and a sodium-independent transporter which does notrequire sodium ion for its function.

However, in a conventional method, it is difficult to analyze thedetails of the transport of an amino acid or its analogue via the acidicamino acid transport system and the in vivo functional roles, and it hasbeen desired to enable a detailed functional analysis by isolating agene of acidic amino acid transporter responsible for the function ofthe acidic amino acid transport system.

With regard to sodium-dependent acidic amino acid transporters, fivekinds of glutamate transporters—EAAC1, GLT-1, GLAST, EAAT4 andEAAT5—have been cloned (Kanai, Curr. Opin. Cell Biol., volume 9, page565, 1997; Kanai and Endou, Curr. Drug Metab., volume 2, page 339,2001).

With regard to sodium-independent transporters, LAT1 (Kanai, et al., J.Biol. Chem., volume 273, pages 23629-23632, 1998) and LAT 2 (Segawa, etal., J. Biol. Chem., volume 274, pages 19745-19751, 1999) have beencloned as neutral amino acid transporters corresponding to a transportsystem L. It was also shown that LAT1 and LAT2 function only when theycoexist with a cofactor 4F2hc which is a single membrane-spanning typeprotein. LAT1 shows an exchange transport activity which transportslarge-sized neutral amino acids such as leucine, isoleucine, valine,phenylalanine, tyrosine, tryptophan, methionine and histidine while LAT2shows a broad substrate selectivity transporting small-sized neutralamino acids such as glycine, alanine, serine, cysteine and threonine inaddition to large-sized neutral amino acids and they are not acidicamino transporters.

With regard to proteins analogous to LAT1 and LAT2, the above-mentionedy⁺LAT1 and y⁺LAT2 having the functions of a transport system y⁺L whichtransports neutral amino acids and basic amino acids have been cloned(Torrents, et al., J. Biol. Chem., volume 273, pages 32437-32445, 1998).It was also revealed that both of y⁺LAT1 and y⁺LAT2 function only whenbeing coexisting with a cofactor 4F2hc. y⁺LAT1 and y⁺LAT2 mainlytransport glutamine, leucine and isoleucine as neutral amino acids anddo not transport acidic amino acids.

With regard to a transporter which requires a cofactor 4F2hc forexpressing its function, Asc-1 which is a protein analogous to LAT1 toLAT2 was cloned (Fukasawa, et al., J. Biol. Chem., 275: 9690-9698,2000). Asc-1 selectively transports alanine, serine, cysteine,threonine, glycine, etc., shows a substrate selectivity of amino acidtransport system asc and does not transport acidic amino acids.

With regard to a transporter which requires another cofactor rBAT havingan analogous structure to 4F2hc for expressing its function, BAT1 whichis a protein analogous to LAT1 and LAT2 was cloned (Chairoungdua, etal., J. Biol. Chem., 274: 28845-28848, 1999). BAT1 transports cystine,neutral amino acids and basic amino acids and does not transport acidicamino acids.

As described above, molecular entity of a transporter which functions bybinding to 4F2hc and rBAT was characterized and, the presence of a groupof transporters which achieves a transport ability by formingheterodimer with a single membrane-spanning type protein and aheterodimeric amino acid transporter family was established.

Further, with regard to a transporter requiring a cofactor 4F2hc forexpressing its function, xCT which is a protein analogous to LAT1 andLAT2 was cloned (Sato, et al., J. Biol. Chem., 274; 11455-11458, 1999).xCT transports cystine, glutamic acid and sodium aminoadipate in asodium-independent manner and corresponds to an amino acid transportsystem Xc. xCT needs a negative charge of side chain of amino acid forrecognition of substrate and is classified under sodium-independentacidic amino acid transporters (Kanai and Endou, Curr. Drug Metab.,volume 2, page 339, 2001).

xCT transports glutamic acid but does not transport aspartic acid andits transport is suppressed by cystine. In addition, xCT is atransporter where expression is induced by oxidative stress and, excepta few cases, its expression in common normal tissues is not detected.However, it has been reported that there is a sodium-independentglutamic acid and aspartic acid transporter which is not suppressed bycystine (Christensen, Physiol. Rev., volume 70, page 43, 1990) and ithas been suggested that there is a sodium-independent acidic amino acidtransporter other than xCT which has not been identified.

Further, Asc-2 which is a protein having an analogous structure to LAT1and LAT2 and binds to unidentified protein other than rBAT or 4F2hc wascloned (Chairoungdua, et al., J. Biol. Chem., 276: 49390-49399, 2001).Asc-2 is not expressed in a cell membrane by itself, however, bypreparing a fusion protein with 4F2hc or rBAT, it transfers to a cellmembrane as a fusion protein and a transport activity can be detected.When Asc-2 is expressed in a cell membrane as a fusion protein with4F2hc or rBAT, it shows a characteristic of a sodium-independent neutralamino acid transport system asc.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a gene of a transporterwhich transports acidic amino acids such as glutamic acid and asparticacid in a sodium-independent manner and also to provide asodium-independent acidic amino acid transporter which is a polypeptideencoded by the gene.

Other objects will be apparent from the following description.

The present inventors have searched the EST (expressed sequence tag)database using a base sequence of translation region of cDNA of BAT1 andidentified a base sequence analogous to BAT1. A base sequence of cDNAclone corresponding to the sequence has been decided and clarified thatit encodes a novel protein. Further, a fusion protein of the translatedproduct of the gene with 4F2hc or rBAT has been prepared and expressedin a cell membrane of oocyte of Xenopus. As a result, it has beenclarified that the function of the translated product of the gene is asodium-independent transporter which transports acidic amino acids suchas glutamic acid and aspartic acid whereby the present invention hasbeen achieved.

Thus, the present invention relates to a protein selected from thefollowing (A) or (B).

(A) protein comprising an amino acid sequence represented by SEQ ID NO:1.

(B) protein comprising an amino acid sequence where one or several aminoacid(s) is/are deleted, substituted or added in the amino acid sequencerepresented by SEQ ID NO: 1 and having an ability of transport of acidicamino acids or its analogue in a sodium-independent manner.

The present invention also relates to a gene comprising DNA selectedfrom the following (a) and (b).

(a) DNA comprising a base sequence represented by SEQ ID NO: 2.

(b) DNA hybridizing with DNA comprising the base sequence represented bySEQ ID NO: 2 under a stringent condition and encodes a protein having anability of transport of acidic amino acids or its analogue in asodium-independent manner.

The novel protein of the present invention having an ability oftransport of acidic amino acids and its analogue in a sodium-independentmanner or, in other words, an amino acid transporter AGT1(aspartate/glutamate transporter 1) is expressed in a cell membrane andhas an ability of transport (uptake) of acidic amino acids such asglutamic acid and aspartic acid in a highly affinitive manner bypreparing a fusion protein with 4F2hc or rBAT.

Incidentally, the sodium-independent transporter AGT1 of the presentinvention which transports acidic amino acids is mainly expressed in thekidney in vivo.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequences of mouse AGT1, mouse Asc-2, ratLAT1, rat y⁺LAT1, mouse xCT and rat BAT1 for comparison with each other.The assumed membrane-spanning sites are shown by lines. A conservedcystine residue is shown by *, assumed cAMP-dependent phosphorylationsite is shown by #, an assumed C-kinase-dependent phosphorylation siteis shown by + and an assumed tyrosine phosphorylation site is shown by&.

FIG. 2 is a photographic picture as a substitute for drawing which showsthe result of analysis of expression of AGT1 gene mRNA in various organtissues of mouse by means of a northern blotting.

FIG. 3 is a photograph as a substitute for drawing which shows theresult of the western blotting analysis by an anti-AGT1 antibody. It wascarried out under a non-reducing condition (−) and a reducing condition(+) in a mouse kidney membrane specimen.

FIG. 4 is a photograph as a substitute for drawing which shows theresult of immunohistological analysis of AGT1 by an anti-AGT1 antibodyin a mouse kidney. a: slightly magnified image. Stainings are observedin proximal tubule of outer layer of medulla and in distal tubule ofcortex. b: absorption experiment by antigen peptide. Stainings observedin “a” disappeared and specificity of staining was shown. c and d:highly magnified images of proximal tubule (c) and distal tubule (d).Stainings are observed in the side of basolateral membrane.

FIG. 5 is a schematic drawing of a fusion protein prepared by connectingAGT1 with 4F2hc or rBAT. Amino acid sequences and gene base sequences ofthe connection parts of AGT1-4F2hc fusion protein and AGT1-rBAT fusionprotein are shown below in FIG. 5.

FIG. 6 shows the result of experiment of the uptake of aspartic acid byan oocyte into which mouse 4F2hc gene cRNA, AGT1 gene cRNA, AGT1 genecRNA/mouse 4F2hc gene cRNA, AGT1-4F2hc fusion protein gene cRNA orAGT1-rBAT fusion protein gene cRNA is injected.

FIG. 7 shows the result of experiment of the uptake of aspartic acid byCOS-7 cells into which mouse 4F2hc gene, mouse rBAT gene, AGT1 gene,AGT1 gene/mouse 4F2hc gene or AGT1 gene/mouse rBAT gene is injected.

FIG. 8 is a drawing which shows the result of investigating theexpression of a fusion protein of AGT1 with 4F2hc (AGT1-4F2hc) in anoocyte cell membrane by a immunofluorescence analysis. As controls,investigations by a immunofluorescence analysis were carried out usingan anti-4F2hc antibody (a, c and e) or an anti-AGT1 antibody in theoocyte into which water was injected (a and b), the oocyte into whichAGT1 gene cRNA was injected and expressed (c and d) and the oocyte intowhich a fusion protein (AGT1-4F2hc) gene cRNA of AGT1 with 4F2hc wasinjected and expressed (e and f).

FIG. 9 shows the result of investigating the influence of added salt inan experiment of the uptake of aspartic acid by an oocyte into which afusion protein (AGT1-4F2hc) gene cRNA of AGT1 with 4F2hc was injected.

FIG. 10 shows the result of investigating the influence of theconcentration of substrate aspartic acid in an experiment of the uptakeof aspartic acid by an oocyte into which a fusion protein (AGT1-4F2hc)gene cRNA of AGT1 with 4F2hc was injected.

FIG. 11 shows the result of investigating the influence of addition ofvarious amino acids and analogous compounds on a system in an experimentof the uptake of aspartic acid by an oocyte into which a fusion protein(AGT1-4F2hc) gene cRNA of AGT1 with 4F2hc was injected.

FIG. 12 shows the result of investigating the influence of addition ofvarious acidic amino acids and analogous compounds on a system in anexperiment of the uptake of aspartic acid by an oocyte into which afusion protein (AGT1-4F2hc) gene cRNA of AGT1 with 4F2hc was injected.

PDC: L-trans-pyrrolidine-2,4-dicarboxylate; DHK: dihydrokainate; **: thecase where p<0.01 in Student's t-test to the data which does notconstitute a pair.

FIG. 13 shows the result of investigating the uptake of radio-labeledamino acid by an oocyte into which a fusion protein (AGT1-4F2hc) genecRNA of AGT1 with 4F2hc was injected.

BEST MODE FOR CARRYING OUT THE INVENTION

SEQ ID NO: 1 in the Sequence Listing which will be shown laterrepresents an amino acid sequence (478 amino acids) of asodium-independent transporter (mouse AGT1) derived from mousetransporting acidic amino acids and SEQ ID NO: 2 represents an aminoacid sequence (478 amino acids) of protein encoded in a full-length cDNAbase sequence (about 2.1 kbp) of the gene and a translation regionthereof.

When the amino acid sequence represented by SEQ ID NO: 1 or the basesequence represented by SEQ ID NO: 2 which will be mentioned later wassubjected to a homology search for all sequences included in the knownprotein databases (NBRF and SWISS-PROT) and DNA databases (GenBank andEMBL), no sequence was identical, whereby the sequence is believed to benovel.

With regard to the protein of the present invention, there may beexemplified, in addition to the one having an amino acid sequencerepresented by SEQ ID NO: 1, a protein having the amino acid sequencewhere one or several amino acid(s) is/are deleted, substituted or addedin the amino acid sequence represented by SEQ ID NO: 1. Deletion,substitution or addition of amino acid(s) may be within such an extentthat transport activity of a neutral amino acid is not lost and,usually, it is from 1 to about 96 or, preferably, from 1 to about 48.Such a protein usually has a homology to the amino acid sequencerepresented by SEQ ID NO: 1 to an extent of 1 to 80%, preferably, 1 to90%.

With regard to the gene of the present invention, in addition to the onehaving a base sequence represented by SEQ ID NO: 2, it includes a genecontaining DNA which can be hybridized with DNA having a base sequencerepresented by SEQ ID NO: 2 under a stringent condition. With regard tothe DNA which can be hybridized, any substance will do so far as theprotein encoded by the DNA has an ability of transporting a neutralamino acid. Such a DNA usually has a homology of base sequence of morethan 70%, preferably more than 80% with the base sequence represented bySEQ ID NO: 2. Such a DNA includes a variant gene found in nature, anartificially modified variant gene, a homologous gene derived from otherorganism, and the like.

In the present invention, a hybridization under a stringent condition isusually carried out in such a manner that a hybridization is carried outfor about 12 hours under the temperature of 37 to 42° C. in ahybridization solution of 5×SSC or having the identical saltconcentration therewith, a preliminary washing is carried out uponnecessity using a solution of 5×SSC or having the identical saltconcentration therewith and then washing is carried out in a solution of1×SSC or having the identical salt concentration therewith.

The sodium-independent transporter gene which transports acidic aminoacids according to the present invention can be isolated and obtained bycarrying out a screening using a tissue or a cell of appropriate mammalsas a gene source. Examples of the mammals are non-human animals such asdog, cattle, horse, goat, sheep, monkey, pig, rabbit, rat and mouse, andin addition to those, human beings.

Screening and isolation of gene is able to be advantageously carried outby, for example, a homology cloning method.

For example, mouse or human kidney is used as a gene source and mRNA(poly(A)⁺RNA) is prepared therefrom. Then a cDNA library is constructedtherefrom and cDNA is screened using a probe corresponding to a sequenceanalogous to BAT1 (such as GenBank™/EBI/DDBJ accession No AI314100)obtained by searching the EST (expressed sequence tag) database wherebya clone containing cDNA of Asc-2 gene can be obtained.

With regard to the obtained cDNA, the base sequence is determined by aconventional method and translation region is analyzed, whereby an aminoacid sequence of the protein encoded thereby, i.e. AGT1, can bedetermined.

The fact that the resulting cDNA is a sodium-independent transporterwhich transports acidic amino acids, in other words, the gene productencoded with cDNA is a sodium-independent transporter which transportsacidic amino acids can be tested, for example, by the following method.Thus, cDNA encoding a fusion protein of AGT1 with 4F2hc or rBAT isprepared using the resulting cDNA of AGT1 gene, then RNA (cRNA) which isprepared from the cDNA and complementary thereto is introduced into theoocyte to be expressed and an ability of transport (uptake) of acidicamino acids into the cells can be confirmed by measuring the uptake ofthe substrate into cells by a common uptake test using an appropriateacidic amino acid as a substrate (Kanai and Hediger, Nature, volume 360,pages 467-471, 1992).

AGT1 protein is synthesized by an in vitro translation method (Hediger,et al., Biochim. Biophys. Acta, volume 1064, page 360, 1991) using RNA(cRNA) prepared from the resulting cDNA of AGT1 gene and beingcomplementary thereto and the size of the protein or the presence ofsugar, etc. can be investigated by means of electrophoresis.

Since cDNA of 4F2hc gene has been reported already (Fukasawa, et al., J.Biol. Chem., 275: 9690-9698, 2000), it is possible to easily prepare agene of 4F2hc from the sequence information by a PCR or the like.

Since cDNA of rBAT gene has been also reported already (Segawa, H., etal., Biochem. J, 328: 657-664, 2000), it is possible to easily prepare agene of rBAT from the sequence information by a PCR or the like.

cDNA encoding a fusion protein of AGT1 with 4F2hc or rBAT is easilyprepared by a PCR or the like from cDNA of AGT1 gene, cDNA of 4F2hc geneor cDNA of rBAT gene.

The characteristic of AGT1 such as substrate selectivity of AGT1 can beinvestigated by applying the similar uptake experiment to the expressedcells.

Homologous gene, chromosome gene, etc. derived from different tissuesand different organisms can be isolated by screening an appropriate cDNAlibrary or genomic DNA library prepared from different gene sourcesusing the resulting cDNA of AGT1 gene.

It is also possible to isolate a gene from a cDNA library or a genomicDNA library by a conventional PCR (polymerase chain reaction) methodusing a synthetic primer designed on the basis of information of thedisclosed base sequence of the gene of the present invention (the basesequence represented by SEQ ID NO: 2 or a part thereof).

DNA library such as a cDNA library or a genomic DNA library may beprepared by a method mentioned, for example, in “Molecular Cloning” bySambrook, J., Fritsh, E. F. and Manitis, T. (Cold Spring Harbor Press,1989). When there is a commercially available library, it can be used aswell.

The sodium-independent transporter which transports acidic amino acidsaccording to the present invention and the gene (AGT1) thereof may beproduced by, for example, a gene recombination technique using cDNAencoding therefor. For example, DNA (such as cDNA) encoding AGT1 isincorporated into an appropriate expression vector and the resultingrecombinant DNA can be introduced into an appropriate host cell. Withregard to an expression system (host-vector system) for the productionof polypeptide, there may be exemplified expression systems of bacteria,yeasts, insect cells and mammalian cells. Among those, it is preferredto use insect cells and mammalian cells for the preparation offunctional proteins.

A fusion protein of the sodium-independent transporter which transportsacidic amino acids according to the present invention with 4F2hc or rBATor a gene thereof (AGT1-4F2hc or AGT1-rBAT) may be produced, forexample, by a gene recombination technique using cDNA encoding it. Forexample, DNA (such as cDNA) encoding AGT1-4F2hc or AGT1-rBAT isincorporated into an appropriate expression vector and the resultingrecombinant DNA can be introduced into an appropriate host cell. Withregard to an expression system (host-vector system) for the productionof polypeptide, there may be exemplified expression systems of bacteria,yeasts, insect cells and mammalian cells. Among those, it is preferredto use insect cells and mammalian cells for the preparation offunctional proteins.

For example, when polypeptide is expressed in mammalian cells, DNAencoding the sodium-independent transporter AGT1 which transports acidicamino acids according to the present invention or DNA encoding a fusionprotein of AGT1 with 4F2hc or rBAT is inserted into a downstream side ofan appropriate promoter (such as cytomegalovirus promoter, SV40promoter, LTR promoter, elongation 1 a promoter, etc.) in an appropriateexpression vector (such as adenovirus vector, retrovirus vector,papilloma virus vector, vaccinia virus vector, SV40 vector, etc.) sothat expression vector is constructed. Then, an appropriate animal cellis transformed using the resulting expression vector and thetransformant is incubated in an appropriate medium whereby a desiredpolypeptide is produced. Examples of the mammalian cell used as a hostare cell strains such as simian COS-7 cell, Chinese hamster CHO cell andhuman HeLa cell.

Accordingly, the present invention provides a vector, preferably anexpression vector, which contains a gene encoding the above-mentionedgene of the present invention or for a protein in the gene and alsoprovides a host cell (transformant) which is transformed using thevector.

With regard to the DNA encoding the sodium-independent transporter AGT1which transports acidic amino acids, cDNA having the base sequencerepresented by SEQ ID NO: 2 may be used, for example, in addition tothat, DNA corresponding to the amino acid sequence is designed and maybe used as DNA encoding polypeptide without limiting to theabove-mentioned cDNA sequence. In that case, with regard to a codonencoding one amino acid, 1 to 6 kinds are known for each, and althoughthe used codon may be optionally selected, frequency of use of a codonof a host utilized for the expression may be taken into consideration todesign a sequence having higher expression efficiency. DNA having thedesigned base sequence can be prepared by chemical synthesis of DNA,binding to fragmentation of the above-mentioned cDNA, partialmodification of the base sequence, and the like. The artificial partialmodification of and introduction of variation into base sequence may becarried out utilizing a primer comprising synthetic oligonucleotideencoding the desired modification by a site-specific mutagenesis (Mark,D. F., et al., Proceedings of National Academy of Sciences, volume 81,page 5662 (1984), etc.).

DNA encoding a fusion protein (AGT1-4F2hc or AGT1-rBAT) of thesodium-independent transporter AGT1 which transports acidic amino acidswith 4F2hc or rBAT may be prepared, for example, using a base sequencerepresented by SEQ ID NO: 2 or SEQ ID NO: 4 or using cDNA having a basesequence represented by SEQ ID NO: 6 and, moreover, DNA corresponding tothe amino acid sequence is designed and can be used as DNA encodingpolypeptide without limiting to the above-mentioned cDNA sequences. Inthat case, with regard to a codon encoding one amino acid, 1 to 6 kindsare known for each, and although the used codon may be optionallyselected, frequency of use of a codon of a host utilized for theexpression may be taken into consideration to design a sequence havinghigher expression efficiency may be designed. DNA having the designedbase sequence can be prepared by chemical synthesis of DNA, binding tofragmentation of the above-mentioned cDNA, partial modification of thebase sequence, and the like. The artificial partial modification andintroduction of variation into base sequence may be carried oututilizing a primer comprising synthetic oligonucleotide encoding thedesired modification by a site-specific mutagenesis (Mark, D. F., etal., Proceedings of National Academy of Sciences, volume 81, page 5662(1984), etc.).

The present invention also provides a nucleotide containing a partialsequence of continuous 14 or more bases, preferably 20 or more bases, inthe base sequence represented by SEQ ID NO: 2 or the complementarysequence thereof.

The nucleotide of the present invention can be used as a probe fordetection of a gene encoding a protein having an ability of transport ofacidic amino acids or its analogue in a sodium-independent manner. Itcan be also used as a primer for obtaining a gene encoding the proteinand the gene encoding a protein having high homology thereto. Further,it can be used for modulation of expression of a gene encoding a proteinhaving an ability to transport acidic amino acids and its analogue in asodium-independent manner by its anti-sense chain, etc.

It is possible to prepare the corresponding antibody using thesodium-independent transporter which transports acidic amino acids ofthe present invention or using a polypeptide having an immunologicalhomology thereto. The antibody can be utilized for detection,purification, and the like. of the sodium-independent transporter whichtransports acidic amino acids. The antibody can be manufactured usingthe sodium-independent transporter which transports acidic amino acidsaccording to the present invention, a fragment thereof, syntheticpeptide having a partial sequence thereof, etc. as an antigen.Polyclonal antibody can be manufactured by a conventional method whereantigen is inoculated to a host animal (such as rat and rabbit) andimmune serum is recovered therefrom while monoclonal antibody can bemanufactured by a conventional technique such as a hybridoma method.

The protein of the present invention has an ability of transportingacidic amino acids and its analogue in a sodium-independently manner andthe ability is strongly affected in the presence of various substances.By screening a substance which inhibits or accelerates the ability, theability of the present protein for transporting the substance can becontrolled.

Accordingly, the present invention provides a method for detecting aneffect of a test substance as a substrate on an ability of the proteinof the present invention for transporting acidic amino acids or itsanalogue in a sodium-independent manner using the above-mentionedprotein of the present invention.

The amino acid which is transported by the protein of the presentinvention is the substance essential for proliferation and growth ofcells and for maintenance of life, and by controlling the uptake of sucha substance into cells, proliferation, growth, etc. of cells can becontrolled. Accordingly, the present invention provides a method forcontrolling the cell proliferation by modulating an ability of theprotein for transporting the acidic amino acids and analogous substancethereto using the above-mentioned protein of the present invention, aspecific antibody thereof or a function-promoting orfunction-suppressing substance thereof.

Gene of a fusion protein of the sodium-independent transporter AGT1transporting the acidic amino acids with 4F2hc or rBAT according to thepresent invention and the expressed cell thereof can be used for an invitro test for the efficiency of permeation of a substance at a cellmembrane where AGT1 is present or at the site where the presence of AGT1is assumed. In addition, a gene of a fusion protein of thesodium-independent transporter AGT1 transporting the acidic amino acidswith 4F2hc or rBAT and the expressed cell thereof can be used for thedevelopment of a compound which efficiently permeates through a cellmembrane where AGT1 is present or at the site where the presence of AGT1is assumed. Further, a gene of a fusion protein of thesodium-independent transporter AGT1 transporting the acidic amino acidswith 4F2hc or rBAT and the expressed cell thereof can be used for an invitro test of pharmaceutical interaction at a cell membrane where AGT1is present or at the site where the presence of AGT1 is assumed.

Accordingly, the present invention provides a method for changing thepharmacokinetics of pharmaceuticals or xenobiotics transported by theabove-mentioned protein of the present invention by using the protein, aspecific antibody thereof or a function-promoting orfunction-suppressing substance thereof, by modulating an ability of theprotein for transport acidic amino acids or its analogue.

As described above, since the protein of the present invention has anability to transport acidic amino acids or its analogue in asodium-independent manner and this ability can be suppressed or promotednot only by the number of a protein existing in a cell, but also by thepresence of various substances (in the presence of afunction-suppressing substance, etc. or in the presence of afunction-promoting substance, etc., respectively), the present inventionprovides a controlling agent for transport ability of a protein foracidic amino acids or its analogue possessed by the above-mentionedprotein of the present invention which comprises the protein, a specificantibody thereof or a function-promoting substance orfunction-suppressing substance thereof.

Since the controlling agent for transport ability of the presentinvention can control the proliferation, growth, and the like. of cells,it can be used as a controlling agent for cell proliferation, and sincethe agent can modulate and control the pharmacokinetics ofpharmaceutical, toxic substance or xenobiotics, it can be used as acontrolling agent for pharmacokinetics of pharmaceutical, toxin orxenobiotics.

By suppressing the sodium-independent transporter AGT1 of the presentinvention which transports acidic amino acids, the permeation of aspecific compound through the cell membrane where AGT1 is expressed orthrough the site where AGT1 is assumed to be present can be limited. Inaddition, a gene of a fusion protein of the sodium-independenttransporter AGT1 of the present invention transporting the acidic aminoacids with 4F2hc or rBAT and its expression cell can be used for thedevelopment of a pharmaceutical (such as a specific inhibitor for AGT1)which limits the permeation of a compound transported by AGT1 through acell membrane or the site where AGT1 is assumed to be present.

Further, in accordance with the present invention, it has been foundthat the protein having an amino acid sequence represented by SEQ ID NO:3 or NO: 5 comprises an ability to promote the transfer AGT1 into a cellmembrane. Accordingly, the present invention provides a promoting agentfor the transfer of AGT1 into a cell membrane containing a proteinhaving an amino acid sequence represented by SEQ ID NO: 3 or NO: 5 or aprotein having an amino acid sequence where one or several amino acid(s)of the above protein is/are deleted, substituted or added.

All of the contents mentioned in the specification of the Japanesepatent application No. 2002-040,608 shall be incorporated into thepresent specification.

EXAMPLES

The present invention will now be illustrated in more detail by way ofthe following Examples although those Examples do not limit the presentinvention.

In the following Examples, each operation was carried out, unlessotherwise clearly mentioned, according to a method mentioned in“Molecular Cloning” by Sambrook, J., Fritsh, E. F. and Manitis, T. (ColdSpring Harbor Press, 1989) or according to the Directions for Use of thecommercially available products when the commercially available reagentsor kits are used.

Example 1

Cloning and expression analysis of a sodium-independent transporterwhich transports acidic amino acids

(1) Identification of Mouse cDNA of a Sodium-Independent Transporterwhich Transports Acidic Amino Acids

cDNA clone corresponding to the base sequence GenBank™/EBI/DDBJaccession No. AI314100 derived from mouse analogous to rat BAT1 obtainedby searching the EST (expressed sequence tag) database using a basesequence of a translation region of rat BAT1 (Chairoungdua, et al., J.Biol. Chem., 274: 28845-28848, 1999) was purchased from IMAGE(Integrated and Molecular Analysis of Genomes and their Expression)(IMAGE clone I. D.: 1907807) and its fragment (1.8-kb) cleaved by arestriction enzyme XhoI was labeled with ³²P-dCTP and used as a probewhereby a mouse kidney cDNA library was screened.

The cDNA library was prepared from poly(A)⁺RNA derived from mouse kidneyusing a kit for the synthesis of cDNA (trade name: Superscript ChoiceSystem, manufactured by Gibco) and incorporated into a site of phagevector AZipLox (manufactured by Gibco) cleaved by a restriction enzymeEcoRI. Hybridization by a probe labeled with ³²P-dCTP was carried outfor one night in a solution for hybridization of 37° C. and the filtermembrane was washed with 0.1×SSC/0.1% SDS at 37° C. With regard to thesolution for hybridization, a buffer of pH 6.5 containing 5×SSC,3×Denhard's solution, 0.2% SDS, 10% dextran sulfate, 50% formamide,0.01% Abtiform B (trade name; Sigma) (antifoaming agent), 0.2 mg/mlsalmon sperm-modified DNA, 2.5 mM sodium pyrophosphate and 25 mM MES wasused. The cDNA portion of λZipLox phage into which cDNA was incorporatedwas incorporated into a plasmid pZL1. The resulting cDNA-insertedfragment of clone was further incorporated into an NotI-cleaved site ofa plasmid pcDNA 3.1 (Invitrogen).

A base sequence for the full-length cDNA was determined by a dyeterminator cycle sequencing method (Applied Biosystems) using asynthetic primer for the determination of base sequence. Further, a basesequence of cDNA was analyzed by a conventional method and translationregion of cDNA and amino acid sequence of the protein encoded therebywere determined.

Those sequences are represented in SEQ ID NO: 1 of the Sequence Listingwhich will be shown later.

AGT1 comprised a 48% homology to a mouse transporter Asc-2 correspondingto a neutral amino acid transport system asc. Further, AGT1 comprised a35% homology to a rat transporter LAT1 and 37% to LAT2 corresponding toa neutral amino acid transport system L, 37% homology to a rattransporter y⁺LAT1 and 36% to a human transporter y⁺LAT2 correspondingto neutral and basic amino acid transport system y⁺L. Furthermore, AGT1comprised a 37% homology to a mouse transporter Asc-1 corresponding to aneutral amino acid transport system asc, 37% to a mouse transporter xCTcorresponding to a cystine and acidic amino acid transport system x, and36% to a rat transporter BAT1 corresponding to a cystine, neutral andbasic amino acid transport system b^(0,+). Still further, Asc-2comprised a 30% homology to a mouse and human transporter CAT1 to 4corresponding to a basic amino acid transport system y⁺.

Comparison of AGT1 with mouse Asc-2, rat LAT1, rat y⁺LAT1, mouse xCT andrat BAT1 in terms of amino acid sequences is shown in FIG. 1.

When an amino acid sequence of AGT1 was analyzed by an SOSUI algorithm(Hirokawa, T., et al., Bioinformatics, volume 14, page 378 (1998)), 12membrane-spanning domains were assumed as shown in FIG. 1. In the thirdhydrophilic loop, conserved cysteine residues were present among Asc-2,LAT1, Asc-2, y⁺LAT1, xCT and BAT1. It is assumed that, via the cysteineresidue, Asc-2 is binded to unknown cofactor via a disulfide bond. Inaddition, there were sites believed to be a cAMP-dependentphosphorylation site in the eighth hydrophilic loop, C-kinase-dependentphosphorylation sites in an N-terminal intracellular region and thesixth hydrophilic loop and a tyrosine phosphorylation site in anN-terminal intracellular region respectively.

(2) Expression of AGT1 Gene in Various Tissues of Mouse (Analysis byNorthern Blotting)

cDNA fragments corresponding to 43rd to 1836th base pair of AGT1 genewere amplified by a PCR, labeled with ³²P-dCTP and using as a probe, anorthern blotting was carried out in the following manner to RNAextracted from various tissues of mouse. 3 μg of poly(A)⁺RNA wassubjected to an electrophoresis using 1% agarose/formaldehyde gel andthen transferred to a nitrocellulose filter. The filter was subjected toa hybridization for one night using a hybridization solution containingAsc-2 cDNA fragments labeled with ³²P-dCTP. The filter was washed at 65°C. with 0.1×SSC containing 0.1% SDS.

As a result of the northern blotting (FIG. 2), a band was detected atabout 2.2 kb in the kidney.

(3) Expression of AGT1 Protein in the Mouse Kidney

A specific antibody to synthetic oligopeptide (CIPDVSDDHIHEES)(mentioned in SEQ ID NO: 7 of Sequence Listing) corresponding to 465-478of mouse AGT1 was prepared according to a method of Altman, et al.(Altman, et al., Proc. Natl. Acad. Sci. USA, volume 81, pages 2176-2180,1984).

Membrane fraction of mouse kidney was prepared according to a method ofThorens, et al. (Thorens, et al., Cell, volume 55, pages 281-290, 1988).The protein sample was treated at 100° C. for 5 minutes in the presence(under a reducing condition) or absence (under a non-reducing condition)of 5% 2-mercaptoethanol, subjected to electrophoresis by anSDS-polyacrylamide gel, blotted to Hybond-P PVDV transfer membrane andtreated with an anti-AGT1 antiserum (1:10,000).

As a result, in the mouse kidney, a band was detected near 250 kDa undera non-reducing condition by an anti-AGT1 antibody as shown in FIG. 3.Under a reducing condition, a band was detected near 40 kDa. From thoseresults, it is suggested that AGT1 is binded to some protein by adisulfide bond.

(4) Immunohistological Analysis of AGT1 Protein in the Mouse Kidney

According to a conventional method, a mouse kidney paraffin slice wastreated with an anti-AGT1 antiserum (1:1,000) and colored withdiaminobenzidine. Further, with an object of investigating thespecificity of color development, an experiment of treating with ananti-AGT1 antiserum (1:1,000) in the presence of 50 mg/ml of an antigenpeptide was also carried out.

As a result, in the mouse kidney, stainings were noted in proximaltubule of outer layer of medulla and in distal tubule of cortex as shownin FIG. 4 a. As the stainings were not detected when an anti-AGT1antiserum was made to act in the presence of an antigen peptide, thespecificity in staining was shown (FIG. 4 b). Further, when anobservation was conducted with highly magnified, it was clarified thatAGT1 protein was present in basolateral membrane of proximal tubule(FIG. 4 c) and distal tubule (FIG. 4 d).

Example 2 Preparation of a Fusion Protein of Sodium-IndependentTransporter AGT1 Transporting Acidic Amino Acids with 4F2hc or with rBATand Analysis of its Function

(1) Preparation of a Fusion Protein of Sodium-Independent TransporterAGT1 Transporting Acidic Amino Acids with 4F2hc or rBAT

In order to prepare a fusion protein of AGT1 with rBAT(AGT1-rBAT), a PCRwas carried out using synthetic oligo-DNA primers5′-GCGCGAAGCTTACCTATAGGCAGAAACATTC-3′ (in which, to a sequencecorresponding to 4th to 23rd base pair of AGT1 cDNA were added asequence corresponding to cleaved site with HindIII and GCGC at 5′-side;mentioned in SEQ ID NO: 8 of the Sequence Listing) and5′-ATATGCGGCCGCACTTTCTTCATGTATGTGGT-3′ (in which, to a sequencecorresponding to 1473rd to 1492nd base pair of AGT1 cDNA were added asequence corresponding to the cleaved site with NotI and ATAT at5′-side; mentioned in SEQ ID NO: 9 of the Sequence Listing) where AGT1cDNA was used as a template. The resulting PCR product was cleaved withHindIII and NotI and ligated to HindIII and NotI sites of mammalian cellexpression vector pcDNA3.1(+) (Invitrogen). Further, a PCR was carriedout using a synthetic oligo-DNA primers5′-ATATGCGGCCGCAGATGAGGACAAAGGCAAGAG-3′ (in which, to a sequencecorresponding to the base pair immediately after translation initiationcodon ATG of mouse rBAT to 21st as shown in SEQ ID NO: 6 were added asequence corresponding to a site cleaved by NotI and ATAT at 5′-side;mentioned in SEQ ID NO: 10 in the Sequence Listing) and5′-GCGCGCTCTAGAAATGCTTTAGTATTTGGCATAATC-3′ (in which, to a sequence of2228th to 2251st base pair of mouse rBAT as shown by SEQ ID NO: 6 wereadded a sequence corresponding to a site cleaved with XbaI and GCGC at5′-side; mentioned in SEQ ID NO: 11 in the Sequence Listing) where rBATcDNA was used as a template. The resulting PCR product was cleaved withNotI and XbaI and ligated to NotI and XbaI sites of the mammalian cellexpression vector pcDNA3.1(+) into which the above-mentioned AGT1 PCRproduct was incorporated to prepare cDNA encoding a fusion protein ofAGT1 with rBAT (FIG. 5).

In order to prepare a fusion protein of AGT1 with 4F2hc(AGT1-4F2hc), aPCR was carried out using synthetic oligo-DNA primers5′-GCGCGAAGCTTACCTATAGGCAGAAACATTC-3′ (in which, to a sequencecorresponding to 4th to 23rd base pair of AGT1 cDNA were added asequence corresponding to a site cleaved by HindIII and GCGC at 5′-side;mentioned in SEQ ID NO: 8 of the Sequence Listing) and5′-ATATGCGGCCGCACTTTCTTCATGTATGTGGT-3′ (in which, to a sequencecorresponding to 1473rd to 1492nd base pair of AGT1 cDNA were added asequence corresponding to a site cleaved by NotI and ATAT at 5′-side;mentioned in SEQ ID NO: 9 of the Sequence Listing) where AGT1 cDNA wasused as a template. The resulting PCR product was cleaved with HindIIIand NotI and ligated to HindIII and NotI sites of mammalian cellexpression vector pcDNA3.1(+) (Invitrogen). Further, a PCR was carriedout using synthetic oligo-DNA primers5′-ATATGCGGCCGCAAGCCAGGACACCGAAGTGGA-3′ (in which, to a sequencecorresponding to the base pair immediately after translation initiationcodon ATG of mouse 4F2hc to 21st as shown in SEQ ID NO: 4 were added asequence corresponding to a site cleaved by NotI and ATAT at 5′-side;mentioned in SEQ ID NO: 12 in the Sequence Listing) and55′-GCGCTCTAGACATGAGGCAGGGGTGATGTTIT-3′ (in which, to a sequencecorresponding to 1820th to 1838th base pair of mouse 4F2hc shown in SEQID NO: 4 were added a sequence corresponding to a site cleaved by XbaIand GCGC at 5′-side; mentioned in SEQ ID NO: 13 of the Sequence Listing)where 4F2hc cDNA was used as a template. The resulting PCR product wascleaved with NotI and XbaI and ligated to NotI and XbaI sites ofmammalian cell expression vector pcDNA3.1(+) into which theabove-mentioned AGT1 PCR product was incorporated to give cDNA encodinga fusion protein of AGT1 with 4F2hc (FIG. 5).

(2) Expression of a Function of a Fusion Protein of a Sodium-IndependentTransporter AGT1 Transporting Acidic Amino Acids with 4F2hc or rBAT

Comparisons were conducted for the uptake of aspartic acid when mouseAGT1 gene cRNA was expressed in the oocyte, when mouse AGT1 gene cRNAand mouse 4F2hc gene cRNA were expressed in the oocyte and when a fusionprotein of ACT1 with 4F2hc or rBAT was expressed in the oocyte.

25 ng of mouse 4F2hc gene cRNA, 25 ng of AGT1 gene cRNA, 12.5 ng of AGT1gene cRNA/12.5 ng of mouse 4F2hc gene cRNA, 25 ng of AGT1-4F2hc fusionprotein gene cRNA or 25 ng of AGT1-rBAT fusion protein gene cRNA wasinjected into the oocyte to express and incubation was conducted for 3days.

With regard to the oocyte into which mouse 4F2hc gene cRNA, AGT1 genecRNA, AGT1 gene cRNA/mouse 4F2hc gene cRNA, AGT1-4F2hc fusion proteingene cRNA or AGT1-rBAT fusion protein gene cRNA was injected,experiments for the uptake of a substrate was carried out using asparticacid as a substrate according to a method of Kanai, et al. (Kanai andHediger, Nature, volume 360, pages 467-471, 1992) as follows. The oocytewere allowed to stand for 30 minutes in a sodium-free uptake solution(100 mM choline chloride, 2 mM potassium chloride, 1.8 mM calciumchloride, 1 mM magnesium chloride and 5 mM HEPES; pH 7.4) containing¹⁴C-aspartic acid (20 mM) as a substrate and an uptake rate of thesubstrate was measured by the count of radioactivity incorporated intothe cells.

As a result (FIG. 6), the levels of the uptake of aspartic acid in theoocyte where only 4F2hc was expressed, the oocyte where only AGT1 wasexpressed and the oocyte where both AGT1 and 4F2hc were co-expressedwere similar to that in the control oocyte into which water wasinjected, while a higher uptake of aspartic acid was noted in the oocytewhere AGT1-rBAT or AGT1-4F2hc was expressed.

It was investigated that rBAT or 4F2hc cannot be a direct cofactor ofAGT1 using COS-7 cells. According to a method mentioned in Mizoguchi, etal., Kidney Int, 59: 1821-1833, 2001, plasmid DNA (each 1 mg) containingAGT1 cDNA, rBAT cDNA or 4F2hc cDNA was introduced into COS-7 cells usingLIPOFECTAMINE 2000 Reagent (Life Technologies). After the introduction,the cells were incubated for two days in a 24-well plate and the uptakeof ¹⁴C-aspartic acid (20 mM) was measured. Measurement of the uptake wasconducted according to a method of Mizoguchi, et al., Kidney Int, 59:1821-1833, 2001, in which it was started by removing the culture liquidand adding Dulbecco's PBS (manufactured by Gibco) containing¹⁴C-aspartic acid, and completed by removing it and washing withice-cooled Dulbecco's PBS. After the washing, it was dissolved with 0.1NNaOH and radioactivity was measured by a liquid scintillation counter.

As a result (FIG. 7), the levels of the uptake of aspartic acid in theoocyte where only 4F2hc was expressed, the oocyte where only rBAT wasexpressed, the oocyte where only AGT1 was expressed, the oocyte whereboth AGT1 and 4F2hc were co-expressed and the oocyte where both AGT1 andrBAT were co-expressed were similar to that the control oocyte intowhich a pcDNA 3.1 plasmid containing no inserted cDNA whereby it wasconfirmed that rBAT or 4F2hc was not a direct cofactor for AGT1.

(3) Identification of Expression of a Fusion Protein ofSodium-Independent Transporter AGT1 Transporting Acidic Amino Acids with4F2hc (AGT1-4F2hc) in Oocyte Cell Membrane by a ImmunofluorescenceAnalysis

Whether the fact that when AGT1 was expressed in the oocyte, no functionwas observed while a fusion protein of AGT1 with 4F2hc (AGT1-4F2hc)showed a functional activity is due to the fact that AGT1 is nottransported to a cell membrane while AGT1-4F2hc is transported to a cellmembrane or not was investigated by a immunofluorescence analysis.

25 ng of AGT1 gene cRNA or 25 ng of the cRNA of a gene of a fusionprotein of AGT1 with 4F2hc(AGT1-4F2hc) was injected into the oocyte toexpress, incubated for 3 days and, the oocyte was fixed in a 4%paraformaldehyde-phosphate buffer and prepared a paraffin section (3 mm)according to a conventional method. After removing the paraffin, thesection was subjected to a blocking with 5% goat serum in 0.05M Trisbuffer in a physiological saline containing 0.1% Tween 20 and treatedwith an affinity-purified anti-Asc-2 antibody or an affinity-purifiedanti-4F2hc antibody (Fukasawa, et al., J. Biol. Chem., 275: 9690-9698,2000). Then, the section was treated with Alexa Fluor 488-labeled goatanti-rabbit IgG (Molecular Probe, Inc.), washed with 0.05M Tris bufferin physiological saline containing 0.1% Tween 20 and observed withOlympus Fluoview (FV500) confocal laser microscope (Olympus). Excitationwas effected with argon laser. at 488 nm and fluorescence from AlexaFluor 488 was detected using a BA505IF filter.

As a result (FIG. 8), in the oocyte in which AGT1 was expressed, an AGT1protein detected in an anti-AGT1 antibody was not present in a cellmembrane but remained inside the cell membrane (FIG. 8 d), while in anoocyte in which a fusion protein of AGT1 with 4F2hc(AGT1-4F2hc) wasexpressed, an AGT1-4F2hc fusion protein expressed in a cell membrane wasdetected in both anti-4F2hc antibody (FIG. 8 e) and anti-AGT1 antibody(FIG. 80. In the control oocyte into which water was injected, nospecific color development by anti-4F2hc antibody (FIG. 8 a) oranti-AGT1 antibody (FIG. 8 b) was observed. Accordingly, it was proventhat the fact that no function was observed when AGT1 was expressed inthe oocyte while a fusion protein of AGT1 with 4F2hc(AGT1-4F2hc) showeda functional activity is due to the fact that AGT1 is not transported toa cell membrane by itself, while its fusion protein with 4F2hc(AGT1-4F2hc) is transported to a cell membrane.

(4) Salt-Dependency of Transport Activity of AGT1

In an uptake experiment of aspartic acid by the oocyte into which a cRNAof a gene of a fusion protein of AGT1 with 4F2hc or with rBAT(AGT1-4F2hc or AGT1-rBAT) was injected, influence of salt added to themedium was investigated.

An uptake experiment of aspartic acid was carried out according to themethod mentioned in the above Example 2(2) using an oocyte into which acRNA of a gene of a fusion protein of AGT1 with 4F2hc or with rBAT(AGT1-4F2hc or AGT1-rBAT) was injected. With regard to the uptakesolution, a standard uptake solution (100 mM of choline chloride wasexchanged with 100 mM sodium chloride) was used instead of a sodium-freeuptake solution when the effect of sodium ion was investigated. Agluconic acid uptake solution (100 mM sodium chloride was exchanged with100 mM sodium gluconate) was used instead of a standard uptake solutionwhen the effect of chlorine ion was investigated.

As a result (FIG. 9), even when extracellular choline was exchanged withsodium or even when extracellular chlorine was exchanged with gluconateion, it did not affect the uptake of aspartic acid at all. From theabove, it was noted that Asc-2 is a transporter which acts independentlyon sodium ion and chlorine ion.

(5) Michaelis-Menten Kinetic Analysis of AGT1

A Michaelis-Menten kinetic analysis of sodium-independent transporterAGT1 which transports acidic amino acids was carried out. TheMichaelis-Menten kinetic analysis was conducted by investigating thechange in the ratio of uptake of aspartic acid by the difference in thesubstrate aspartic acid concentration.

The aspartic acid uptake experiment was carried according to the methodmentioned in the above Example 2(2) using the oocyte into which a cRNAof a gene of a fusion protein of AGT1 with 4F2hc or with rBAT(AGT1-4F2hc or AGT1-rBAT) was injected. As a result (FIG. 10), the Kmvalue of aspartic acid transport by AGT1-4F2hc was 25.5+5.9 mM (meanvalue+standard error). The Km value of aspartic acid transport byAGT1-rBAT was 20.1+6.1.

The Michaelis-Menten kinetic analysis was similarly carried out in afusion protein of AGT1 with 4F2hc (AGT1-4F2hc) in glutamic acid and Kmvalue and Vmax value were calculated. Result of the above is shown inthe following Table 1.

Table 1

Km values and Vmax values of substrate amino acid

Amino Acid Km^(a) (μM) Vmax^(b) L-Aspartic acid 25.5 ± 5.9 (1.00)L-Glutamic acid 21.8 ± 6.5 0.63 ± 0.10 ^(a, b)V max value of L-glutamicacid is shown by the ratio to Vmax value of L-aspartic acid. Both Km andVmax values are represented by mean value ± standard error.

(6) Substrate Selectivity of AGT1 (Inhibition Experiment Using AddedAmino Acid and Its Analogue)

In an uptake experiment of aspartic acid by an oocyte into which a cRNAof a gene of a fusion protein of AGT1 with 4F2hc (AGT1-4F2hc) wasinjected, the effect of addition of various amino acids and theiranalogues on the system was investigated.

An aspartic acid uptake experiment was carried out according to themethod mentioned in the above Example 2(2) using an oocyte into which acRNA of a gene of a fusion protein of AGT1 with 4F2hc (AGT1-4F2hc) wasinjected. However, a sodium-free uptake solution was used and the uptakeof ¹⁴C-aspartic acid (20 mM) was measured in the presence and absence of2 mM of various compounds (non-labeled).

As a result (FIG. 11), in aspartic acid, glutamic acid and cysteine, asignificant cis-inhibiting effect was observed.

Basic amino acids, neutral amino acids except cysteine, cystine,2-amino-2-norbornane-carboxylic acid (BCH) which is a transport systemL-specific inhibitor, γ-aminoisobutyric acid, α-aminomethylisobutyricacid, D-aspartic acid and D-glutamic acid did no affect the uptake of¹⁴C-aspartic acid mediated by AGT1-4F2hc (FIG. 11).

In an oocyte into which cRNA of a gene of a fusion protein of AGT1 withrBAT (AGT1-rBAT) were injected together, the effect of adding variousamino acids and their analogues on the system was also investigated inan aspartic acid uptake experiment by an oocyte in a similar manner asin the case of AGT1-4F2hc.

As a result, in the case of AGT1-rBAT, the same result as in the case ofAGT1-4F2hc was obtained, and in the case of a fusion protein of AGT1with 4F2hc or with rBAT (AGT1-4F2hc or AGT1-rBAT), the 4F2hc or rBATmoiety did not affect the characteristic of substrate-binding site and,with regard to the information concerning the substrate selectivityobtained in a fusion protein, the AGT1 itself also reflects thetransport characteristic.

In the substances analogous to acidic amino acids,threo-β-hydroxyaspartate (THA), L-serine-O-sulfate (SOS), L-cysteinesulfate and L-cysteate strongly inhibited the uptake of ¹⁴C-asparticacid mediated by AGT1.

On the contrary, in the case of L-α-aminoadipate, L-homocysteate,L-trans-pyrrolidine-2,4-dicarboxylate (PDC) and dihydrokainate (DHK), noinhibition effect on the uptake of ¹⁴C-aspartic acid mediated by AGT1was observed (FIG. 12).

(7) Substrate Selectivity of AGT1 (Uptake Experiment Using Various AminoAcids and their Analogues as Substrates)

Various kinds of amino acids and their analogues were used as substratesand uptake by an oocyte into which a cRNA of a gene of a fusion proteinof AGT1 with 4F2hc (AGT1-4F2hc) was injected was investigated.

The uptake experiment of various amino acids and their analogues wascarried out according to the method mentioned in the above-mentionedExample 2(2) using an oocyte into which a cRNA of a gene of a fusionprotein of AGT1 with 4F2hc (AGT1-4F2hc) was injected. With regard to asubstrate, various compounds which were labeled with radioactivity wereused instead of ¹⁴C-aspartic acid.

As a result, when L-glutamic acid (14C compound) was used as a substratein addition to L-aspartic acid (14C compound) (FIG. 13), a substantialuptake into the oocyte was observed.

INDUSTRIAL APPLICABILITY

The sodium-independent transporter of the present invention whichtransports acidic amino acids and a gene thereof enables an in vitroinvestigation of transport of acidic amino acids and amino acidanalogues including xenobiotics at the site where the transporter isexpressed, and based on which, also enables an in vitro assumption ofpharmacokinetic of these compounds. Further, the present invention isuseful for the development of pharmaceutical which permeates efficientlythrough a site where the transporter is expressed. Furthermore, bymodulating an ability to transport acidic amino acids and its analoguepossessed by the transporter, the invention can be utilized for thedevelopment of a method for controlling a cell proliferation.

1-17. (canceled)
 18. An antibody directed to a protein selected from thegroup consisting of the following (A) and (B): (A) a protein consistingof the amino acid sequence represented by SEQ ID NO: 1; and (B) aprotein having the ability to transport acidic amino acids and theiranalogues in a sodium-independent manner which consists of an amino acidsequence where one or several amino acids are deleted, substituted oradded in the amino acid sequence represented by SEQ ID NO:
 1. 19-28.(canceled)
 29. The antibody of claim 18, wherein the protein is derivedfrom mouse.
 30. The antibody of claim 18, wherein the protein is derivedfrom organ, tissue or cultured cell.
 31. An antibody directed to aprotein selected from the group consisting of the following (C) and (D):(C) a fusion protein of a protein consisting of the amino acid sequencerepresented by SEQ ID NO: 1 and a protein consisting of the amino acidsequence represented by SEQ ID NO: 3; and, (D) a protein having theability to transport acidic amino acids and their analogues in asodium-independent manner which consists of an amino acid sequence whereone or several amino acids are deleted, substituted or added in theamino acid sequence of a fusion protein of a protein consisting of theamino acid sequence represented by SEQ ID NO: 1 and a protein consistingof the amino acid sequence represented by SEQ ID NO:
 3. 32. The antibodyof claim 31, wherein the protein is derived from mouse.
 33. The antibodyof claim 31, wherein the protein is derived from organ, tissue orcultured cell.
 34. An antibody directed to a protein selected from thegroup consisting of the following (E) and (F): (E) a fusion protein of aprotein consisting of the amino acid sequence represented by SEQ ID NO:1 and a protein consisting of the amino acid sequence represented by SEQID NO: 5; and, (F) a protein having the ability to transport acidicamino acids and their analogues in a sodium-independent manner whichconsists of an amino acid sequence where one or several amino acids aredeleted, substituted or added and in the amino acid sequence of a fusionprotein of a protein consisting of the amino acid sequence representedby SEQ ID NO: 1 and a protein consisting of the amino acid sequencerepresented by SEQ ID NO:
 5. 35. The antibody of claim 34, wherein theprotein is derived from mouse.
 36. The antibody of claim 34, wherein theprotein is derived from organ, tissue or cultured cell.